EFNA3, a key functional mediator of hypoxic microenvironment in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of all cancer-related deaths. Its rapid proliferative nature and HCC specific therapy such as transarterial chemoembolization (TACE) that block arterial blood supply to the tumor frequently induce hypoxic microenvironments i.e. regions with oxygen deprivation. Hypoxia has emerged as a major driving force to promote aggressive phenotypes and stemness of cancer cells in solid tumors. We aimed to identify the key contributors to HCC progression in hypoxic microenvironment. From the analysis of transcriptomic profiles of human HCCs in both our in-house RNA-sequencing (RNA-seq) data and TCGA data as well as HCC cells treated with hypoxia and normoxia, we identified Ephrin-A3 (EFNA3) as a potential hypoxia-inducible oncogene. EFNA3 is a membrane-bound ligand which can activate multiple receptor tyrosine kinases of the Eph family. This family of ligands and receptors are frequently dysregulated in multiple cancer types and have implications in stem cell plasticity. We further validated that EFNA3 was significantly overexpressed in HCCs as compared with their non-tumorous liver samples using qRT-PCR in a separate cohort of HCCs (p<0.0001, n=97). Upon clinicopathological correlation, overexpression of EFNA3 was significantly correlated with the presence of venous invasion (p=0.004) and more advanced tumor stage (p=0.026). In concordance, TCGA-LIHC dataset revealed significantly higher expression of EFNA3 in HCC samples with vascular invasion (p=0.0094) and advanced tumor stage (p=0.0002). Furthermore, we confirmed the upregulation of EFNA3 upon hypoxia treatment in multiple HCC cell lines. Functionally, stable knockdown of EFNA3 using short hairpin RNA approach significantly reduced proliferation and migratory rates of HCC cells. In addition, orthotopic liver injection model of the EFNA3 knockdown HCC cells demonstrated a significant reduction of the growth of the primary tumors. There was also a reduced incidence of distant metastasis to lungs in vivo. Using sphere formation assay to test self-renewal ability in vitro, EFNA3-knockdown HCC cells showed significantly reduced self-renewal ability. In addition, HCC cells positive/high for stemness markers (EpCAM, CD13, CD24, CD44, CD47, CD133) isolated from our PDTX model showed a consistently higher EFNA3 mRNA expression as compared with the negative/low cells. By flow cytometric analysis, the EFNA3 KD MHCC-97L demonstrated a decreased expression of CD47. Conclusion: We identified EFNA3 as a potential hypoxia-inducible oncogene in HCC and demonstrated its critical role in tumor progression, metastasis and cancer stemness of HCC. Based on our results, EFNA3 may be an attractive therapeutic target to counteract hypoxia-specific disease progression. Citation Format: Abdullah Husain, Yung Tuen Chiu, Daniel Wai Ho, Karen Man Sze, Lo Kong Chan, Yu Man Tsui, Carmen Chak Wong, Irene Oi Ng. EFNA3, a key functional mediator of hypoxic microenvironment in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2431.